1. Field of the Invention
This invention relates to the fields of medicine and cell biology. More specifically, it relates to the role of certain T-type calcium channels in pain signaling. In particular, it relates to inhibitors of USP5, and their use in controlling pain.
2. Related Art
Low-voltage activated (LVA) T-type calcium channels are essential contributors to signalling in electrically excitable cells. The vertebrate genome encodes three different types of T-type calcium channels (termed Cav3.1, Cav3.2 and Cav3.3, respectively) with specific expression patterns and unique functional and pharmacological profiles (Catterall and Few, 2008; Iftinca et al., 2009; Chen et al., 2003; Andreasen et al., 2000; Bourinet et al., 2005a; Bourinet et al., 2005b; Mangoni et al., 2006; McKay et al., 2006; Perez-Reyes et al., 2003). In addition, alternate splice isoforms of each of the Cav3 channel subtypes have been reported, and have been shown to give rise to channels with distinct functional properties (Catterall and Few, 2008; McKay et al., 2006; Molineux et al., 2006. At the molecular level, T-type channels are formed by a single pore that is comprised of four transmembrane domains that are connected by large cytoplasmic linkers. These linker regions form important protein interaction sites (Iftinca et al., 2011) and are the targets of second messenger systems such as protein kinases and G proteins (Iftinca et al., 2009).
In neurons, T-type channels regulate excitability and neuronal firing patterns, they activate calcium dependent signalling cascades, they contribute to low threshold exocytosis (Weiss et al., 2012), and they are pharmacological targets in neurological disorders such as epilepsy and pain. Indeed, the processing of pain signals via the afferent pain pathway is critically dependent on the activity of T-type calcium channels which not only shape the firing patterns of pain sensing neurons, but also contribute to the release of neurotransmitters at dorsal horn synapses (Jacus et al., 2012). Primary afferent pain fibers have their cell bodies in the dorsal root ganglia (DRG) and express predominantly the Cav3.2 T-type calcium channel subtype, with largest expression levels occurring in medium sized cells (Bourinet et al., 2005b). Genetic ablation of T-type channels, their knockdown via intrathecal delivery of antisense oligonucleotides, or their direct inhibition by small organic molecules results in an increased threshold of both mechanical and thermal pain thus validating T-type channels as a suitable pharmacological target (Bourinet et al., 2005a). T-type channel expression (and thus activity) in pain sensing neurons is upregulated in various pathological conditions linked to pain, including diabetic neuropathy, bowel inflammation, and nerve injury (Marger et al., 2011; Jagodic et al., 2007; Messinger et al., 2009), however, the molecular mechanisms by which this upregulation occurs are unknown. Nonetheless, it stands to reason that preventing this type of enhancement would be a suitable means of combating the development of pain, while sparing the normal function of these channels in other tissues such as the brain and the cardiovascular system.
One possible mechanism by which ion channel expression levels can be regulated is the ubiquitination and proteasomal degradation of these channels. Indeed, there is a large body of evidence in the literature that describes the ubiquitination of ion channels such as CFTR, ENaC and high voltage activated calcium channels by E3 ubiquitin ligases. Akin to the relationship between protein kinases and phosphatases, ubiquitin specific proteases (USP's) or deubiquitinases (DUB's) serve to remove ubiquitin groups from target proteins, leading to an increase in protein stability. However, their role in ion channel stability has been virtually unexplored.